PROJECT SUMMARY/ABSTRACT Autism Spectrum Disorder (ASD) affects millions of individuals in the United States, and is highly costly to society (11,12). In recent years, research on the functional importance of many of ASD-linked genes suggest that components of Neuronal Homeostatic Plasticity are important (1). Homeostatic plasticity provides negative feedback to stabilize neuronal function and works in opposition to destabilizing learning- and experience- associated plasticity. While homeostatic plasticity has been well described, which and how homeostatic mechanisms are altered in ASD remains unknown. This proposal aims to determine which homeostatic mechanisms are altered in ASD and how those alterations affect single neuron and network stability. I hypothesize that ASD-linked mutations affect both homeostasis of neuronal synapses and excitability, giving rise to unstable neural networks ultimately responsible for subserving sensory processing and behavior. I will utilize our model of Timothy Syndrome, a syndromic form of ASD (33,34). In vitro preparations enable me to precisely dissect the mechanisms involved, while in vivo investigations will determine if these altered mechanisms are at play in the animal. I have identified that homeostatic plasticity in neurons expressing the TiS mutation is exaggerated. By chronically blocking action potentials in dissociated cortical cultures for 24hrs with tetrodotoxin (TTX), neurons globally increase their excitatory synaptic weights and their excitability. In TiS neurons, these increases are exaggerated beyond the levels of neurons without the TiS mutation. In Aims 1 & 2, I will determine which molecular mechanisms are involved. Specifically, through a series of electrophysiology and molecular biology experiments, Aim 1 will investigate the role of calcium- permeable AMPARs in mediating exaggerated synaptic homeostasis. Aim 2 will identify what underlying molecular processes are involved in the exaggeration of neuronal firing and excitability in TiS neurons. I will determine whether exaggeration of excitability is mediated by axon initial segment regulation, or by phosphorylation of particular ion channels. I predict that altered regulation of these mechanisms are responsible for functional exaggeration of homeostasis. Furthermore, I hypothesize that this exaggeration in neurons results in maladaptive homeostatic plasticity for networks. In Aim 3, I will describe how the TiS mutation affects neural network activity using electrophysiology and calcium imaging. With pharmacological manipulations, I will dissect which of the previously described homeostatic mechanisms are responsible. I will also determine if maladaptive homeostasis in TiS results in tonic asynchronous or coordinated hypersynchronous activity. The results of this study will elucidate the underlying pathophysiological mechanisms of ASD and may provide a generalizable framework for distinguishing ASD subtypes.